Field of the Invention
The present invention belongs to the field of compliance markers and marker vaccines which allow for the differentiation between infected and vaccinated individuals. In particular, it relates to a compliance marker for vaccines including a subunit antigen, and a DIVA (Differentiating Infected from Vaccinated Animals) system which makes it possible to differentiate between animals infected with a pathogen and animals treated with a subunit antigen derived from said pathogen, wherein said subunit antigen has been expressed in cultured insect cells, preferably by means of a genetically engineered baculovirus.
Description of the Related Art
Baculoviruses are large rod-shaped double stranded DNA viruses that infect invertebrates, in particular insects, but do not replicate in mammalian or other vertebrate cells. Starting in the 1940's they were first used as biopesticides in the crop fields. Additionally, after the publication of a first paper describing the overexpression of human beta interferon in insect cells (Smith et al. Mol Cell Biol. 3: 2156-2165 (1983)), genetically engineered baculoviruses have been widely used for producing complex recombinant proteins in insect cell cultures, including the production of antigens for several approved human and veterinary subunit vaccines (van Oers et al. J Gen Virol. 96: 6-23 (2015)).
Vaccination is an essential tool to manage herd health, in particular in high density confinement settings where many food animals are raised. When disease outbreaks occur in animals that were supposedly vaccinated, questions arise as to whether the vaccine failed to protect the animals or whether the vaccine was delivered properly, wherein the latter possibility regarding proper delivery of the vaccine is referred to as vaccine compliance.
The use of compliance markers for determining if an animal has been properly vaccinated is thus highly desired by producers. WO 2009/058835 A1 describes e.g. the use of purified xylanase which was added as a compliance marker to a swine influenza vaccine. Regarding vaccines comprising baculovirus-expressed subunit antigens, it is possible to use baculovirus antigens as a compliance marker, wherein, however, currently used systems have limitations in animals testing positive and that a high amount of antigen is needed (Gerber et al. Res Vet Sci. 95:775-81 (2013); Lehnert. Top Agrar 5: S11-S14 (2011)).
Vaccines used in programs for controlling viral outbreaks and infections must have an effective system to monitor for continued presence of viral infection within the population. However, vaccination complicates large scale surveillance for the spread of the infection by e.g. serological means, as both vaccinated and exposed individuals produce antibody specific for the virus. The antigenic similarity between the infecting virulent field strain of the virus and the viral vaccine frequently hampers the discrimination between infected and vaccinated subjects as vaccination results in the occurrence and persistence of antibodies that are indistinguishable between infected and vaccinated individuals.
There is increasing worldwide interest in DIVA (differentiating infected and vaccinated animals) vaccination strategies. For example, the joint WHO/FAO/OIE meetings on avian influenza strain H5N1 HPAI have recommended that all vaccination is practiced using a DIVA, so spread of infection can be monitored. However, current DIVA methods are difficult to scale-up and often have problems with the differentiation of vaccination from infection with other circulating viral strains.
Current methods of monitoring include physical tagging of vaccinated animals, the use of sentinel animals, and virological testing. However, these current methods have a number of limitations due to logistical and economic reasons.
The physical tagging of vaccinated animals involves the time consuming individual identification of vaccinated individuals by physical means such as ear tags, leg bands or wing tags. Also, the use of unvaccinated sentinel animals is logistically and economically difficult and there is also a risk that if sentinels become infected with the virus, e.g. poultry infected with H5N1 virus, there is increased risk of spread to humans. Virological testing of individuals via screening and detection of live virus or RT-PCR surveillance testing is a very expensive and infrastructure heavy process, which is not applicable for subunit vaccines, and only provides information relating to the current status of an individual, and does not allow analysis of the infection and/or vaccination history of that individual.
In view of said limitations, the use of marker vaccines allowing a serological discrimination of vaccinated and infected animals is highly preferable, wherein such marker vaccines can be prepared either as negative or positive marker vaccine.
A negative marker vaccine is prepared by using an antigenic portion of the pathogen or by the removal of an antigen from the pathogen, which provokes specific antibodies in infected animals. Negative marker vaccines are usually either subunit vaccines or attenuated live vaccines containing a genetically engineered strain lacking an immunogenic antigen. An example for a negative marker vaccine is e.g. the use of baculovirus-expressed classical swine fever virus (CSFV) E2 protein as a subunit antigen for vaccinating against classical swine fever, wherein a detection of antibodies specific for other antigens of CSFV, e.g. ERNS protein or NS3 protein, in sera of vaccinated pigs shows a CSFV infection.
A positive marker vaccine contains an additional antigen which induces specific antibodies in vaccinated individuals but not in infected ones. An example for a positive marker vaccine approach is described in WO 2007/053899 A1, where inactivated H6N2 Avian Influenza (AI) virus and tetanus toxin, both of which separately produced, were combined in one injection for vaccinating birds, and subsequently antibodies specific for tetanus toxin were detected in sera obtained from said birds as markers showing that the birds were vaccinated.
However, the separate production of both the vaccine antigen and the marker antigen is relatively expensive and, furthermore, a mixing step is required for combining both components in one vaccinating agent, wherein this additionally may also affect the stability of the vaccine components/antigens.
Thus, a simple marker system is needed for inexpensively producing positive marker vaccines, in particular subunit vaccines comprising baculovirus-expressed antigens.
Furthermore, effective compliance markers are needed which also enable the sensitive identification of vaccinations with a low amount of baculovirus-expressed subunit antigen.